Carbohydrate structures present on human tumor cells have been associated with cancer invasion and metastasis (Dennis et. al., Science 236: 582, 1987; Demetriou et al., J. Cell Biol. 130:383, 1995). These structures include the GlcNAc .beta.(T1-6) branched N- and O-linked carbohydrate side chains of cell-surface glycoproteins. The Golgi enzymes required for their synthesis are .beta.(T1-6) N-acetylglucosaminyltransferase V (i.e. GlcNAc-TV) and core 2 .beta.(T1-6) N-acetylglucosaminyltransferase (i.e. core 2 GlcNAc-T), respectively. These enzymes are up-regulated in human carcinomas (Fernandes et al., Cancer Res. 51:718-723, 1991), a phenomenon that has been associated with the activation of the ras signaling pathway (Dennis et al., Science 236:582-585, 1987; Dennis et al Oncogene 4:853-860, 1989)). Furthermore, overexpression of GlcNAc-TV in epithelial cells results in morphological transformation and tumor formation in mice (Demetriou et al, J. Cell Biol. 130:383-392, 1995). Therefore, GlcNAc-TV as well as enzymes supplying acceptor substrates to GlcNAc-TV (i.e. GlcNAc-TI, .alpha.-mannosidase II and core 2 GlcNAc-T of the O-linked pathway) are targets for anti-cancer pharmaceuticals.
Although there are no inhibitors of GlcNAc-TV presently available, a lead .alpha.-mannosidase II inhibitor, swainsonine has been tested in preclinical and human trials. Swainsonine is an indolizidine alkaloid found in Australian Swainsona canescens (Colegate et al., Aust J Chem 32:2257-2264, 1979), North American plants of the genera Astragalus and Oxytropis (Molyneux R J and James L F., Science 215:190-191, 1981), and also the fungus Rhizoctonia leguminicola (Schneider et al., Tetrahedron 39;29-31, 1983). Swainsonine's ability to inhibit .alpha.-mannosidase II activity appears to be responsible for its interesting immunomodulating and cancer suppression activity. Swainsonine is believed to function as an enzyme inhibitor because it can mimic the glycosylium cation intermediate generated during the hydrolytic cleavage of mannopyranosides. (Goss, P. E. et al., Clin. Cancer Res. 1: 935-944, 1995).
The swainsonine blockage of .alpha.-mannosidase II is prior to GlcNAc-TV and prevents expression of GlcNAc .beta.(T1-6) branched N-linked carbohydrates. Swainsonine-treated murine tumor cells have been found to be less metastatic in both organ-colonization and spontaneous metastasis assays in mice (Dennis J. W., Cancer Res. 46:5131-5136, 1986 and Humphries et al., Proc. Natl. Acad. Sci. U.S.A. 83:1752-1756, 1986). Swainsonine has also been shown to block tumor cell invasion through extracellular matrix in vitro (Yegel et al., Int. J. Cancer 44:685-690, 1989 and Seftor et al., Melanoma Res. 1:53-54, 1991). Swainsonine administered either orally or by mini-osmotic pumps to athymic nude mice inhibited the growth rate of human MeWo melanoma and HT29m colon carcinoma tumor xenografts in the mice (Dennis et al., J. Natl. Cancer Inst. 81:1028-1033, 1989 and Dennis et al., Cancer Res., 50:1867-1872, 1990).
Phase I clinical trials of swainsonine have been done which indicate that it has efficacy in the treatment of human tumors. (Goss et. al, Cancer Res., 54:1450, 1995). Although side-effects in humans are mild, some of these may be associated with swainsonine's inhibition of lysosomal storage of carbohydrates.
Swainsonine has positive effects on cellular immunity in mice (reviewed in Humphries M. J. and Olden K., Pharmacol Ther. 44:85-105, 1989, and Olden et al., Pharmacol Ther 50:285-290, 1991)). In particular, swainsonine has been shown to alleviate both chemically-induced and tumor-associated immune suppression (Hino et al., J. Antibiot. (Tokyo) 38:926-935, 1985), increase NK cell (Humphries et al., Cancer Res. 48:1410-1415, 1988), and LAK cell activities (Yagita M and Saksela E., Scand. J. Immunol. 31:275-282, 1990), and increase splenic and bone marrow (BM) cell proliferation (White et al., Biochem. Biophys. Res. Commun. 150;615-625, 1988; Bowlin et al. Cancer Res 49, 4109-4113, 1989, and White et al., Cancer Commun. 3:83-91, 1991). SW has also been shown to be hemorestorative in mice following treatment with both cycle-specific and nonspecific chemotherapeutic agents (Oredipe et al., J. Natl. Cancer Inst. 83:1149-1156, 1991).
Japanese Patent Application No. J61277685 describes indolizidine derivatives which are reported to be useful as immune regulators, which can be administered orally or parenterally at a dose of about 0.1-100 ml/kg a day. It is also reported that the indolizidine derivatives may be used in combination with antitumour agents, antimicrobial agents or antiinflammatories.
Carbonoyloxy substitutions at the 2 and 8 carbons of swainsonine have been reported to reduce inhibitor activity by 2-3 orders of magnitude for Jack Bean and MDAY-D2 tumor cell lysosomal mannosidases in vitro. However, 2-p-nitrobenzoyloxy, 2-octanoyloxy- and 2-butanoyloxy-derivatives of swainsonine retained full activity as inhibitors of Golgi oligosaccharide processing in viable MDAY-D2 tumor cells. Inhibition of oligosaccharide processing was reduced by the esterase inhibitor diethyl p-nitrophenyl phosphate, suggesting that while the compounds are relatively poor inhibitors of mannosidase in vitro, the compounds enter cells at a rate comparable to that of swainsonine and are converted to swainsonine by cellular esterases. The more lipophilic esters, 2-benzoyloxy-swainsonine, 2-toluoyloxy-swainsonine, 8-palmitoyloxy-swainsonine and 8-myristinoyloxy-swainsonine, showed IC.sub.50 values at least 10 times higher for inhibition of Golgi oligosaccharide processing, probably due to less efficient entry of the compounds into tumor cells. The anti-metastatic activities of swainsonine and two analogs were tested and shown to correlate with the IC.sub.50 values for inhibition of Golgi oligosaccharide processing in cultured tumor cells. In vivo, SW and the analogues were administered intraperitoneally to mice and found to have comparable activities as stimulators of bone marrow cell proliferation. (Dennis, J. W. et al. Biochemical Pharmacology 46:1459-1466, 1993).
Selected swainsonine analogues, in particular 2-substituted analogues, and methods for preparing the analogues are described in U.S. Pat. No. 5,466,809.